肠上皮屏障功能丧失是消化道炎症的一个标志。由于屏障研究中缺乏合适的基于细胞的模型，详细机制仍不清楚。在这里，我们对不同条件下的人类肠道类器官培养物进行了详细的功能表征，旨在提出一种优化的离体模型，以进一步分析炎症引起的肠上皮屏障功能障碍。分化的 Caco2 细胞作为肠上皮屏障研究的传统模型，显示出成熟的屏障功能，这些功能在用细胞混合物（TNFα、IFN-γ、IL-1ß）模拟炎症条件后会降低。在培养基中生长的人肠道类器官具有高度增殖性，显示出高水平的 LGR5，但细胞间粘附率总体较低，且屏障功能不成熟，类似于肠隐窝中常见的情况。 WNT 耗竭导致肠道类器官分化，LGR5 水平降低，并且代表沿隐窝绒毛轴存在的所有细胞类型的标记物上调。这与规则分布在细胞边界的连接蛋白的屏障成熟相平行。在未成熟的人肠道类器官培养物中应用细胞混合物会导致屏障功能降低，并伴有细胞破碎、细胞死亡和连接蛋白的整体损失，这表明类器官培养物对炎症刺激具有高度敏感性。在分化的类器官培养物中，细胞混合物诱导了一系列分层变化，从细胞粘附丧失、细胞边界连接蛋白重新分布、蛋白质降解（伴随上皮屏障功能丧失）开始。观察到细胞活力随着时间的推移而降低，但当初始屏障变化明显时细胞活力得以保留。总之，分化的肠道类器官培养物代表了一种优化的人类离体模型，可以全面反映在肠道炎症患者中观察到的情况。我们的数据表明，炎症引起的肠道屏障功能障碍存在一个分层序列，首先是细胞间粘附力丧失，然后是连接蛋白的重新分布和丧失，导致屏障功能降低，并导致上皮细胞连续死亡。版权所有 © 2023 Kollmann、Buerkert、Meir、Richter、Kretzschmar 、弗莱明、凯尔姆、格尔默、奥托、布卡德和施莱格尔。

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.Copyright © 2023 Kollmann, Buerkert, Meir, Richter, Kretzschmar, Flemming, Kelm, Germer, Otto, Burkard and Schlegel.